There are a number of infectious diseases in mammalian subjects that appear to be associated with mis-folded forms of native protein—i.e., are characterized by high levels of normal and abnormal prion protein (PrP). Among these are bovine spongiform encephalopathy (BSE) in bovine subjects, scrapie in sheep, Chronic Wasting Disease (CWD) in ungulates, and Creutzfeldt-Jakob disease (CJD) in humans. Collectively, these conditions are called transmissible spongiform encephalopathies (TSE's) and this terminology will be used in the present application.
The infectious agent for these conditions has not been identified, but it is known that the infection can be transmitted through the food supply as well as through peripheral routes including white blood cells in experimental animals and in humans. Manuelidis, E. E., et al., Science (1978) 200:1069-1071; Manuelidis, E. E., et al., Lancet (1985) 2:896-897. Two strains of CJD have been identified—an attenuated SY strain and a more virulent FU strain. Manuelidis, L., et al., Proc. Natl. Acad. Sci. USA (2003) 100:5360-5365. The ability of the SY strain to protect against infection by the FU strain was ascribed to a successful immune response (ibid.). It has been shown that the infectious particle for CJD has a viral size of about 25 nm and infectivity is markedly reduced by conditions that disrupt viral core components. Manuelidis, L., Viral Immunol. (2003) 16:123-139. Myeloid cells are believed to play a significant role in the spread of infection (ibid.). Long nucleic acids have also been identified in some high titer preparations of the infectious strain (ibid.). See also Manuelidis, L., et al., Neurosci. Lett. (2000) 293:163-166.
The effect of FU and SY strains in neuronal cell lines with differing patters of PrP resistant to proteolysis (PrP-res) was also studied by Arjona, A., et al., Proc. Natl. Acad. Sci. USA (2004) 101:8768-8773. It has also been shown that CJD is transferred from brain via the blood to the gut at early stages of infection. Radebold, K., et al., BMC Infectious Diseases (2001) 1:20. Other animals besides humans, sheep and cattle have also been affected by the same or analogous agents, including pigs, rodents, primates, cats, and various zoo animals. See, for example, Manuelidis, L., et al., Science (1997) 277:94-98.
Earlier work by the present applicants has shown that certain genes are highly expressed in microglia comprised of a specialized type of brain myeloid cells isolated from CJD infected brains. Baker, C. A., et al., J. Virol. (2002) 76:10905-10913 and Baker, C. A., et al., Proc. Natl. Acad. Sci. USA (2003) 100:675-679 isolated microglia cells from the brains of both normal mice and mice infected with a strain of CJD about 130 days after inoculation (after symptoms appear) and determined comparative levels of gene expression using mRNA isolated from the samples and gene microarray technology. It was found that mRNA's involved in inflammatory functions were substantially increased by 5-20 fold in CJD microglia; these included IL1 molecules responsible for macrophage respiratory bursts (gp91phox and p22phox) and several other leukocyte surface molecules, as well as complement cascade components C1q, properdin, and factor H. Also elevated was the complement receptor subunit CD18, lipoprotein lipase, CD36, and CD68. Also elevated was serum amyloid A3 (SAA3) and molecules associated with interferon signaling. The most potently induced transcript was lysozyme M. Extensive profiles were provided in both articles and compared to profiles of expression obtained when normal cells were stimulated with lipopolysaccharide to mimic bacterial infection or by interferon γ to mimic inflammation. Alterations were found in the profiles of normal cells thus stimulated, but these profiles did not replicate those obtained from cells isolated from rodents that had been inoculated with CJD and exhibited symptoms of this condition. Similarly, treating normal cells with prion protein resistant to proteolysis (PrP-res) did not appear to produce the type of changes in expression levels shown in cells derived from CJD inoculated animals.
More recently, Baker, C. A., et al., J Neurovirol. (2004) 10:29-40 further investigated certain interferon-sensitive transcripts as upregulated in brain and in cells of the microglia obtained as soon as 10 days after inoculation. Additional studies of early markers were reported by Lu, Z. Y., et al., J. Cell. Biochem. (2004) 93:644-652.
In addition, Harrison, J. K., et al., Proc. Natl. Acad. Sci. USA (1998) 95:10896-10901 showed that elevated levels of CX3CR1 is associated with mediation of microglial-neuronal interactions; Hashimoto, S. I., et al., Blood (2000) 96:2206-2214 showed that the protease inhibitor cystatin F is specifically expressed in human activated and mature dendritic cells.
It is known that abnormal PrP is formed in the brain about 90 days after infection, i.e., experimental inoculation with rodent-passed CJD or similar CJD strains. Thus, at the time the markers enumerated above were determined, the abnormal PrP presence in the brain was well established. The markers described in the present invention are present at detectable levels before the appearance of abnormal PrP. Furthermore, the markers appear not only in the microglia, but also in the peripheral system, including blood, gut, urine and the like, and in cells isolated from these sources. Thus, these markers provide a convenient early warning assay for TSE infection prior to the appearance of alternative symptomologies, including the appearance of PrP in the microglia. Because these markers are present in the peripheral system, the assessment can be made on accessible body fluids and cells. Further, because some of the markers are different from those in Alzheimer's disease brain, they can be used to distinguish TSE infection from Alzheimer's in symptomatic patients. In addition, use of the markers may distinguish various TSE strains.